The disclosed compounds generally relate to the field of dithio reagents. In particular, the disclosed compounds generally relate to quenched fluorescent dithio reagents that are linked to a peptide via a linker containing a dithio group. The compounds may be useful for detecting thiol-containing compounds and dithio-containing compounds.
Thiols are ubiquitous in cellular biochemistry, playing important roles in determining protein structure (as disulfide linkages) and enzymatic mechanisms (as covalent catalysts). Furthermore, the redox state in the cell is largely regulated by the thiol/disulfide status of glutathione in the cell (i.e., GSH vs GSSG). In addition, reduced glutathione (i.e., the thiol form or GSH) also plays a control role in drug metabolism by attacking electrophilic atoms. Therefore, thiol detection and quantitation is important, in cellular biochemistry, and to date has been accomplished most commonly by performing UV-Visible assays using colon metric reagents such as Ellman's reagent. Recently, new probes and associated methods to detect thiols have also been reported whereby quenched fluorophores are reacted with thiols, thereby leading to a fluorescence signal (e.g., Tang et al. (2007) J. Am. Chem. Soc. 129, 11666; Lin et al. (2009) Chemistry 15, 5096). These methods do not involve use of disulfide probes, especially those tethered to peptides which provide lower background signal relative to other fluorescent disulfide compositions. Furthermore, no probes or methods have been reported to date that permit direct detection of disulfides, especially disulfides inside live cells.
Fluorescence detection systems (e.g., fluorescence spectroscopy) have been widely used to study the structure, mechanism and function of different proteins and enzymes, and especially in enzymatic activity or binding assays. Fluorescence detection systems are useful in that they generally have high sensitivity and a good dynamic range for detection. In addition, many generic fluorescent, reagents are available, as well as commercially available equipment for detecting particular reagents. Fluorescence detection systems may be amenable to high throughput screening (e.g., using any bench-top fluorescence plate reader). Potential drawbacks associated with some fluorescence reagents may include photobleaching, stability, and purity of the utilized fluorophore. Some commercially available fluorescent labeling reagents are mixtures of isomers or have high photobleaching or cause uncontrolled labeling, which prevents them from giving reliable and reproducible results. Some also have background fluorescent signal, and others suffer from having a lack of selectivity for the molecule or functional group (e.g., thiols or disulfides) being detected.
As such, fluorescent dithio reagents are desirable. In particular, fluorescent dithio reagents that are photostable, single isomers are desirable. Further, fluorescent dithio reagents that may be used as cellular probes are desirable.